DESCRIPTION (from applicant's abstract): Synapses throughout the brain are ensheathed by glial cells, which provide nutrients and help to terminate the action of neurotransmitters. It is not known whether glia also actively regulate the development or function of synapses. We have developed methods for the isolation and culture of highly purified populations of CNS neurons and glia. We are using these methods to ask two questions: Do synapses between neurons form in the absence of glial cells? Do glial cells regulate synaptic transmission? Our preliminary findings demonstrate that ultrastructurally normal synapses form in the absence of glia. However, in the absence of glia, several proteins necessary for vesicular release fail to accumulate in presynaptic terminals. Physiological studies and FM1-43 imaging indicate that there is also a very low presynaptic efficacy with little vesicle recycling occurring in response to presynaptic depolarization. Thus, in the absence of glia, synapses form that are biochemically and functionally immature. Glial cells secrete a protein that enhances synaptic protein accumulation and increases presynaptic efficacy by more than ten-fold, as shown by an increase in the frequency of miniature excitatory postsynaptic currents, evoked transmitter release, and vesicle recycling using FM1-43. Taken together, these preliminary findings indicate that glial cells strongly promote presynaptic maturation. In this proposal, we will ask: (1) By what type of biochemical mechanism do glia enhance presynaptic efficacy? (2) By what functional mechanism do glial cells enhance presynaptic efficacy? (3) Do glial cells enhance presynaptic calcium currents? (4) Which synaptic proteins are elevated by glial cells? and (5) What is the identity of the glial-derived protein that enhances presynaptic efficacy? Methods that we will use to answer these questions include patch-clamp recording, immunostaining, FM1-43 imaging, semiquantitative Western Blotting, gene chip profiling, biochemical purification and expression cloning. We will continue to use a culture system that allows highly purified retinal ganglion cells (RGCs) to be maintained for extended periods of culture under serum-free conditions in the presence or absence of glia. RGCs serve as a good model system for CNS glutamatergic neurons and are presently the only defined CNS neuron type that can be highly purified and cultured in the absence of glial cells with high survival. Our ultimate goal is to understand whether glial cells normally regulate synaptic function in vivo. If so, glial regulation of synaptic function may participate in learning and memory or be perturbed by reactive gliosis after brain injury.